Enhanced silk exsertion under stress

ABSTRACT

The invention provides methods for enhancing maize silk exsertion under stress conditions and compositions relating to such methods, including nucleic acids and proteins. The invention further provides recombinant expression cassettes, host cells, and transgenic plants.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 10/409,701 filed Apr. 8, 2003 which claims the benefit of, andincorporates by reference, Provisional Patent Application 60/370,796,filed Apr. 8, 2002.

TECHNICAL FIELD

The present invention relates generally to plant molecular biology. Morespecifically, it relates to nucleic acids and methods for modulatingtheir expression in plants.

BACKGROUND OF THE INVENTION

Throughout their lives, plants are routinely subjected to a variety ofstresses which act to impede or alter growth and development processes.Stress to the growth and development of agricultural plants has anegative economic impact in the form of reduced yields, increasedexpenditures to ameliorate the effects of stress, or both. Given theworld's increasing human population and the diminishing land areaavailable for agriculture, improving agricultural productivity is ofparamount importance. Thus, there is a need for crop plants that arebetter able to tolerate stresses and maintain productivity underunfavorable conditions.

While traditional plant breeding approaches will continue to beimportant for improving agricultural plants, the new strategies that arelikely to have the most significant impact on crop improvement willinvolve genetic engineering. A thorough understanding of the molecularand cellular mechanisms used by plants to avoid or tolerate stresseswill aid in the development of new strategies to improve the stresstolerance of agricultural plants.

Stresses to plants may be caused by both biotic and abiotic agents. Forexample, biotic causes of stress include infection with a pathogen,insect feeding, parasitism by another plant such as mistletoe, andgrazing by animals. Abiotic stresses include, for example, excessive orinsufficient available water, insufficient light intensity, temperatureextremes, synthetic chemicals such as herbicides, and excessive wind.Yet plants survive and often flourish, even under unfavorableconditions, using a variety of internal and external mechanisms foravoiding or tolerating stress. Plants' physiological responses to stressreflect changes in gene expression.

Grain yield in Zea mays is dependent upon the number of ovaries whichare initiated, are fertilized, and develop to maturity. Reduced grainproduction may result from, inter alia, a decrease in the number ofkernel initials, restricted or untimely silk exsertion, and/or kernelabortion during grain development.

Maize silks comprise the stigmatic tissues of the flower, interceptingair-borne pollen and supporting pollen tube growth to result infertilization. Silk receptivity to pollen is limited in duration and isaffected by environmental factors. For example, under droughtconditions, silk exsertion is delayed or restricted and thus may notoccur at the proper time relative to pollen shed. (See, for example,Herrero and Johnson, (1981) Crop Science 21:105-110) Importantly, theprocess of fertilization determines kernel number and thus sets anirreversible upper limit on grain yield.

What is needed in the art is a means to stabilize yield of maize acrossenvironments by ensuring ample and timely silk exsertion. This can beaccomplished through transgenic modifications to create a plant withconstant or increased rates of silk exsertion, even under stress,relative to an unmodified plant.

Modification of gene expression affecting silk growth and developmentrequires use of promoters expressed exclusively or preferentially insilk tissues; for example, see, U.S. Pat. No. 6,515,204. Also needed arecoding regions capable of enhancing silk growth and development. Thepresent invention meets these and other objectives.

SUMMARY OF THE INVENTION

Generally, it is an object of the present invention to provide methodsof transforming plants with genetic constructs comprising novelcombinations of appropriate promoter sequences and coding sequences toresult in transformed plants with improved silk development underconditions of stress, relative to an untransformed plant under stress.Further objects of the present invention are to provide said transgenicplants exhibiting improved silk development under stress, and to providesaid genetic constructs.

For example, cell division may be limiting to silk development understress. Transformation with cytokinin biosynthetic genes would help tocontinue driving cell division in spite of stress to the plant.Arabidopsis plants transformed with ipt from Agrobacterium tumefaciensdemonstrated increased flooding tolerance correlated with increasedexpression of ipt. (VanToai, et al., Abstract P518, Plant and AnimalGenome VII Conference, San Diego, Calif., Jan. 17-21, 1999)Seed-specific expression of the Agrobacterium tumefaciens tzs gene intransgenic tobacco resulted in increased seed weight and number.(Roeckel, et al., (1998) Physiologia Plantarum 102:243-249) Dietrich, etal., (Plant Physiol. Biochem. 33(3):327-336, 1995) showed that maximumcell division activity within developing maize endosperm coincided withthe peak in total kernel cytokinin concentration. Thus, increasingcytokinin levels in the developing silk could serve to increase ormaintain cell division and silk exsertion under stress conditions.

Alternatively or additionally, cell cycle genes, such as cyclin D, wouldhelp to continue driving cell division and thus maintain silkdevelopment in spite of stress to the plant. Cockcroft, et al., (Nature405:575-579, 2000) found that tobacco plants transformed with CycD2under control of a constitutive promoter had elevated overall growthrates. Riou-Khamlichi, et al., (Science 283:1541-1544, 1999) reportedthat CycD3 could induce cell division when constitutively expressed intransgenic Arabidopsis callus. Thus, increased expression of cell cyclegenes specifically in silk tissue could promote cell division and silkgrowth.

Alternatively or additionally, transformation resulting in increased,directed expression of sucrose symporters could increase the carbonsupply to developing silks. Symporters act to accumulate sucrose fromthe apoplast and transport it across cell boundaries, such as intophloem sieve elements or companion cells. Symporters may also transportmonosaccharides, and their activity could be important in silkdevelopment, providing hexoses to elongating cells to maintain osmoticpressure and provide precursors for macromolecular synthesis. For areview, see, Williams, et al., (2000) Trends in Plant Science5(7):283-290. There is evidence for tissue specificity and fortranscriptional regulation of expression of sucrose symporters(Williams, supra, at pp. 287 and 289). Further, biotic and abioticstresses can affect the expression of sucrose symporters. See, forexample, Noiraud, et al., (2000) Plant Physiology 122:1447-1455. Thus,constructs directing increased or sustained expression of sucrosesymporters in female reproductive tissues at critical developmentalstages could be useful in maintaining growth and function of the silks.Leggewie, et al., (U.S. Pat. No. 6,025,544) teach transformation withsucrose transporter sequences for earlier and/or more prolificflowering. The present invention, in contrast, provides transformationwith sucrose transporter sequences to result in sustained or improvedsilk development under conditions of stress, especially drought, densityand/or heat stress.

Alternatively or additionally, transformation resulting in targetedupregulation of invertases could increase the carbon supply todeveloping silks. Invertases convert sucrose into its componentmonosaccharides, glucose and fructose. Soluble invertase activitycreating an increased solute concentration within a cell would serve todraw water into the cell and cause it to expand. In maize, a solubleinvertase, Ivr2, has been shown to be specifically induced under waterstress, and the resulting increase in hexose accumulation was speculatedto increase osmotic pressure which could provide drought resistance.(Kim, et al., (2000) Plant Physiology 124:71-84). Overexpression of Ivr2in silk tissues could therefore drive desirable cell expansion underconditions of water stress.

Alternatively or additionally, increased expression of a sodiumantiporter within silk tissues could result in improved silk developmentunder drought stress. Overexpression in Brassica napus of AtNHX1,encoding a vacuolar sodium antiporter from Arabidopsis, produces plantswith reduced sensitivity to salt. Sodium ions are moved into thevacuole, increasing the solute concentration, which results in waterbeing drawn into the cell. (Zhang, et al., (2001) PNAS98(22):12832-12836) Overexpression of AtNHX1 or a gene encoding aprotein of similar function within maize silk tissues could thereforedrive increased water rentention and desirable cell expansion underconditions of water stress. Such gene may be from maize.

Alternatively or additionally, adequate osmotic potential for cellexpansion could result from directed overexpression of a vacuolarpyrophosphatase. Gaxiola, et al., have reported that overexpression ofthe Arabidopsis AVP1 gene, which encodes a vacuolar pyrophosphatase,resulted in marked drought tolerance, apparently through increasedvacuolar accumulation of solute causing enhanced cellular waterretention. (PNAS 98(20):11444-11449, 2001). Overexpression of AVP1 or agene encoding a protein of similar function within maize silk tissuescould therefore drive increased water retention and desirable cellexpansion under conditions of water stress.

Alternatively or additionally, expansins could help to drive silk cellexpansion. Expansins are extracellular proteins which catalyze cell-wallenlargement by breaking non-covalent bonds between cell-wallpolysaccharides. Increased expression of expansin genes has beencorrelated with rapid stem growth in submerged rice (Cho and Kende,(1997) Plant Cell 9:1661-1671) and with root growth of maize seedlingsunder drought stress (Wu, et al., (1996) Plant Physiol. 111:765-772).Directed expression of expansins could aid in cell enlargement, thusincreasing silk length, particularly under stress conditions.

Alternatively or additionally, directed expression of aquaporins couldaid in silk cell expansion. Aquaporins, designated TIPs (TonoplastIntrinsic Proteins) or MIPs (plasma-Membrane Intrinsic Proteins), arechannel proteins which facilitate water movement across vacuolar orplasma membranes. The maize aquaporin gene ZmTIP1 is expressed at highlevels in expanding cells, consistent with the hypothesis that TIPsallow rapid uptake of water. (Chaumont, et al., (1998) Plant Physiol.117:1143-1152) Upregulated and directed expression of aquaporins,including those endogenous to maize and particularly those expressed inmaize silk tissue, could support rapid silk cell expansion and thuspromote silk tissue growth. See, also, U.S. Pat. Nos. 6,313,375 and6,313,376, hereby incorporated by reference.

Alternatively or additionally, targeted expression of genes encodingenzymes involved in raffinose synthase may provide tolerance of drought,salinity, and/or cold. Taji, et al., (Plant Journal 29(4):417-426, 2002)have reported that “stress-inducible galactinol synthase plays a keyrole in the accumulation of galactinol and raffinose under abioticstress conditions” and that “galactinol and raffinose may function asosmoprotectants in drought-stress tolerance of plants.” Therefore,constructs directing overexpression of galactinol synthase or raffinosesynthase in silk tissue could lead to improved silk exsertion underabiotic stress.

It is a further object of the invention to provide promoter sequencesactive exclusively or preferentially in silks and methods of use of thepromoter sequences. In other aspects the present invention relatesto: 1) recombinant expression cassettes, comprising a nucleic acid ofthe present invention operably linked to a promoter, 2) a host cell intowhich has been introduced the recombinant expression cassette, and 3) atransgenic plant comprising the recombinant expression cassette. Thehost cell and plant are optionally from maize.

It is a further object of the present invention to provide a method ofimproved control of expression of an endogenous or exogenous product ina transformed plant or its progeny.

It is a further object of the present invention to provide a method foreffecting useful changes in the phenotype of a transformed plant or itsprogeny.

It is a further object of the present invention to provide a method formodulating the development of a transformed plant or its progeny.

In a further aspect, the present invention relates to a method formodulating gene expression in a stably transformed plant comprising thesteps of (a) transforming a plant cell with a recombinant expressioncassette of the present invention; (b) growing the plant cell underappropriate growing conditions and (c) regenerating a stably transformedplant from the plant cell wherein said linked nucleotide sequence isexpressed.

DETAILED DESCRIPTION OF THE INVENTION

Overview

A. Nucleic Acids and Proteins of the Present Invention

Unless otherwise stated, the polynucleotide and polypeptide sequencesidentified in SEQ ID NOS: 1-26 represent exemplary polynucleotides andpolypeptides useful in the present invention. Table 1 providesidentification of SEQ ID NOS: 1-26.

Polynucleotide Polypeptide Gene Name SEQ ID NO. SEQ ID NO.Silk-preferred promoter (“gl2”) 1, 26 — Isopentenyl transferase 2 3Cyclin D 4 5 Cyclin-dependent kinase 6 7 α-expansin 8 9 β-expansin 10 11Aquaporin 12 13 Aquaporin 14 15 Aquaporin 16 17 Sucrose symporter 18 19Soluble invertase 20 21 Sodium antiporter 22 23 Vacuolar pyrophosphatase24 25B. Exemplary Utility of the Present Invention

The present invention provides utility in such exemplary applications asengineering Zea mays plants to exhibit improved silk exsertion, relativeto a non-transformed plant, under conditions of environmental stress,such as drought, high plant density, or excessive heat.

Improved silk exsertion may comprise elements of timeliness and quality,for example, more rapid exsertion, greater silk length, and morecomplete and/or more uniform silk emergence from the ear shoot. Suchimprovements in silk exsertion may result from, for example, increasedrates of cell division in silk tissue, increased expansion of cellscomposing silks, and altered rates of flow of water and solutes withinor into silk tissue.

Definitions

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges recitedwithin the specification are inclusive of the numbers defining the rangeand include each integer within the defined range. Amino acids may bereferred to herein by either their commonly known three-letter symbolsor by the one-letter symbols recommended by the IUPAC-IUBMB NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. Unless otherwise provided for, software,electrical, and electronics terms as used herein are as defined in TheNew IEEE Standard Dictionary of Electrical and Electronics Terms (5^(th)edition, 1993). The terms defined below are more fully defined byreference to the specification as a whole. Section headings providedthroughout the specification are not limitations to the various objectsand embodiments of the present invention.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, Persing, et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

As used herein, “antisense orientation” includes reference to a duplexpolynucleotide sequence that is operably linked to a promoter in anorientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise interveningsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening sequences (e.g., as in cDNA). Theinformation by which a protein is encoded is specified by the use ofcodons. Typically, the amino acid sequence is encoded by the nucleicacid using the “universal” genetic code. However, variants of theuniversal code, such as are present in some plant, animal, and fungalmitochondria, the bacterium Mycoplasma capricolum, or the ciliateMacronucleus, may be used when the nucleic acid is expressed therein.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host organism.For example, although nucleic acid sequences of the present inventionmay be expressed in both monocotyledonous and dicotyledonous plantspecies, sequences can be modified to account for the specific codonpreferences and GC content preferences of monocotyledons or dicotyledonsas these preferences have been shown to differ (Murray, et al., (1989)Nucl. Acids Res. 17: 477-498). Thus, the maize-preferred codon for aparticular amino acid may be derived from known gene sequences frommaize. Maize codon usage for 28 genes from maize plants is listed inTable 4 of Murray, et al., supra.

As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of a native (non-synthetic), endogenous, biologically (e.g.,structurally or catalytically) active form of the specified protein.Methods to determine whether a sequence is full-length are well known inthe art, including such exemplary techniques as northern or westernblots, primer extension, S1 protection, and ribonuclease protection.See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Comparison to known full-lengthhomologous (orthologous and/or paralogous) sequences can also be used toidentify full-length sequences of the present invention. Additionally,consensus sequences typically present at the 5′ and 3′ untranslatedregions of mRNA aid in the identification of a polynucleotide asfull-length. For example, the consensus sequence ANNNNAUGG, where theunderlined codon represents the N-terminal methionine, aids indetermining whether the polynucleotide has a complete 5′ end. Consensussequences at the 3′ end, such as polyadenylation sequences, aid indetermining whether the polynucleotide has a complete 3′ end.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by human intervention. For example, apromoter operably linked to a heterologous structural gene is from aspecies different from that from which the structural gene was derived,or, if from the same species, one or both are substantially modifiedfrom their original form. A heterologous protein may originate from aforeign species or, if from the same species, is substantially modifiedfrom its original form by human intervention.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “introduced” includes reference to the incorporation of anucleic acid into a eukaryotic or prokaryotic cell where the nucleicacid may be incorporated into the genome of the cell (e.g., chromosome,plasmid, plastid or mitochondrial DNA), converted into an autonomousreplicon, or transiently expressed (e.g., transfected mRNA). The termincludes such nucleic acid introduction means as “transfection”,“transformation” and “transduction”.

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is substantially free from components that normallyaccompany or interact with it in its naturally-occurring environment.The isolated material optionally comprises material not found with thematerial in its natural environment, or if the material is in itsnatural environment, the material has been synthetically (non-naturally)altered by human intervention to a composition and/or placed at alocation in the cell (e.g., genome or subcellular organelle) not nativeto the isolated material. The alteration to yield the synthetic materialcan be performed on the material within or removed from its naturalstate. For example, a naturally-occurring nucleic acid becomes anisolated nucleic acid if it is altered, or if it is transcribed from DNAwhich has been altered, by means of human intervention performed withinthe cell from which it originates. See, e.g., Compounds and Methods forSite Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No.5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells;Zarling, et al., PCT/US93/03868. Likewise, a naturally-occurring nucleicacid (e.g., a promoter) becomes isolated if it is introduced bynon-naturally-occurring means to a locus of the genome not native tothat nucleic acid.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer, or chimeras thereof, ineither single- or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to that of naturally-occurring nucleotides (e.g., peptidenucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism, or of a tissueor cell type from that organism. Construction of exemplary nucleic acidlibraries, such as genomic and cDNA libraries, is taught in standardmolecular biology references such as Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol. 152, AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook, et al., MolecularCloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and CurrentProtocols in Molecular Biology, Ausubel, et al., Eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (1994).

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. Plant cell, as used herein includes, withoutlimitation, cells isolated from seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores. The class of plantswhich can be used in the methods of the invention include bothmonocotyledonous and dicotyledonous plants. A particularly preferredplant is Zea mays.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or chimeras or analogsthereof that have the essential nature of a natural deoxy- orribo-nucleotide in that they hybridize, under stringent hybridizationconditions, to substantially the same nucleotide sequence as donaturally-occurring nucleotides and/or allow translation into the sameamino acid(s) as do the naturally-occurring nucleotide(s). Apolynucleotide can be full-length or a subsequence of a native orheterologous structural or regulatory gene. Unless otherwise indicated,the term includes reference to the specified sequence as well as to thecomplementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to naturally-occurring amino acid polymers, as well as toamino acid polymers in which one or more amino acid residue is anartificial chemical analogue of a corresponding naturally-occurringamino acid. The essential nature of such analogues ofnaturally-occurring amino acids is that, when incorporated into aprotein, that protein is specifically reactive to antibodies elicited tothe same protein but consisting entirely of naturally-occurring aminoacids. The terms “polypeptide”, “peptide” and “protein” are alsoinclusive of modifications including, but not limited to, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation. Further, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of proteins of the invention.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses, and bacteria which comprise genes expressed inplant cells such Agrobacterium or Rhizobium. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, or seeds. Suchpromoters are referred to as “tissue preferred”. Promoters whichinitiate transcription only in certain tissue are referred to as “tissuespecific”. A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “repressible” promoter is apromoter which is under environmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue specific, tissuepreferred, cell type specific, and inducible promoters constitute theclass of “non-constitutive” promoters. A “constitutive” promoter is apromoter which is active under most conditions.

As used herein “recombinant” includes reference to a cell or vector thathas been modified by the introduction of a heterologous nucleic acid orto a cell derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found in identical formwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under-expressed or notexpressed at all, as a result of human intervention. The term“recombinant” as used herein does not encompass the alteration of thecell or vector by naturally-occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a promoter and a nucleic acid to betranscribed.

The terms “residue” and “amino acid residue” and “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally-occurring amino acid and, unless otherwiselimited, may encompass non-natural analogs of natural amino acids thatcan function in a similar manner as naturally-occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, preferably 90% sequenceidentity, and most preferably 100% sequence identity (i.e., arecomplementary) with each other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, (1984) Anal. Biochem., 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. Hybridizationand/or wash conditions can be applied for at least 10, 30, 60, 90, 120or 240 minutes. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

As used herein, “transgenic plant” includes reference to a plant whichcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally-occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used inintroduction of a polynucleotide of the present invention into a hostcell. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween a polynucleotide/polypeptide of the present invention with areference polynucleotide/polypeptide: (a) “reference sequence”, (b)“comparison window”, (c) “sequence identity”, and (d) “percentage ofsequence identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison with a polynucleotide/polypeptide of thepresent invention. A reference sequence may be a subset or the entiretyof a specified sequence; for example, as a segment of a full-length cDNAor gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” includes reference to acontiguous and specified segment of a polynucleotide/polypeptidesequence, wherein the polynucleotide/polypeptide sequence may becompared to a reference sequence and wherein the portion of thepolynucleotide/polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides/amino acids residues in length, andoptionally can be 30, 40, 50, 100, 200, 300, 400, 500, 600, 750, 1000,1250, 1500 or longer. Those of skill in the art understand that to avoida high similarity to a reference sequence due to inclusion of gaps inthe polynucleotide/polypeptide sequence, a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, (1981) Adv. Appl.Math. 2:482; by the homology alignment algorithm of Needleman andWunsch, (1970) J. Mol. Biol. 48:443; by the search for similarity methodof Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2444; bycomputerized implementations of these programs, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA. The CLUSTAL program is well described byHiggins and Sharp, (1988) Gene 73:237-244; Higgins and Sharp, (1989)CABIOS 5:151-153; Corpet, et al., (1988) Nucleic Acids Research16:10881-90; Huang, et al., (1992) Computer Applications in theBiosciences 8:155-65, and Pearson, (1994) et al., Methods in MolecularBiology 24:307-331.

The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995); Altschul, et al.,(1990) J. Mol. Biol. 215:403-410; and Altschul, et al., (1997) NucleicAcids Res. 25:3389-3402.

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information. This programinvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold. These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always>0) and N (penalty score for mismatching residues;always<0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST program parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see, Henikoff and Henikoff, (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLAST programalso performs a statistical analysis of the similarity between twosequences (see, e.g., Karlin and Altschul, (1993) Proc. Natl. Acad. Sci.USA 90:5873-5877). One measure of similarity provided by the BLASTprogram is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences.However, many real proteins comprise regions of nonrandom sequenceswhich may be homopolymeric tracts, short-period repeats, or regionsenriched in one or more amino acids. Such low-complexity regions may bealigned between unrelated proteins even though other regions of theprotein are entirely dissimilar. A number of low-complexity filterprograms can be employed to reduce such low-complexity alignments. Forexample, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-163)and XNU (Clayerie and States, (1993) Comput. Chem. 17:191-201)low-complexity filters can be employed alone or in combination.

Unless otherwise stated, nucleotide and protein identity/similarityvalues provided herein are calculated using GAP (GCG Version 10) underdefault values.

GAP (Global Alignment Program) can also be used to compare apolynucleotide or polypeptide of the present invention with a referencesequence. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.48:443-453, 1970) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. For polypeptide sequences thedefault gap creation penalty is 8 while the default gap extensionpenalty is 2. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 100. Thus, for example, the gap creation and gap extensionpenalties can each independently be: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl.Acad. Sci. USA 89:10915).

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins and Sharp, (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci.,4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

Utilities

The present invention provides, among other things, compositions andmethods for modulating (i.e., increasing or decreasing) the level ofpolynucleotides and polypeptides of the present invention in plants. Inparticular, the polynucleotides and polypeptides of the presentinvention can be expressed temporally or spatially, e.g., atdevelopmental stages, in tissues, and/or in quantities, which areuncharacteristic of non-recombinantly engineered plants.

The present invention also provides isolated nucleic acids comprisingpolynucleotides of sufficient length and complementarity to apolynucleotide of the present invention to use as probes oramplification primers in the detection, quantitation, or isolation ofgene transcripts. For example, isolated nucleic acids of the presentinvention can be used as probes in detecting deficiencies in the levelof mRNA in screenings for desired transgenic plants, for detectingmutations in the gene (e.g., substitutions, deletions, or additions),for monitoring upregulation of expression or changes in enzyme activityin screening assays of compounds, for detection of any number of allelicvariants (polymorphisms), orthologs, or paralogs of the gene, or forsite directed mutagenesis in eukaryotic cells (see, e.g., U.S. Pat. No.5,565,350). The isolated nucleic acids of the present invention can alsobe used for recombinant expression of their encoded polypeptides, or foruse as immunogens in the preparation and/or screening of antibodies. Theisolated nucleic acids of the present invention can also be employed foruse in sense or antisense suppression of one or more genes of thepresent invention in a host cell, tissue, or plant. Attachment ofchemical agents which bind, intercalate, cleave and/or crosslink to theisolated nucleic acids of the present invention can also be used tomodulate transcription or translation.

The present invention also provides isolated proteins comprising apolypeptide of the present invention (e.g., preproenzyme, proenzyme, orenzymes). The present invention also provides proteins comprising atleast one epitope from a polypeptide of the present invention. Theproteins of the present invention can be employed in assays for enzymeagonists or antagonists of enzyme function, or for use as immunogens orantigens to obtain antibodies specifically immunoreactive with a proteinof the present invention. Such antibodies can be used in assays forexpression levels, for identifying and/or isolating nucleic acids of thepresent invention from expression libraries, for identification ofhomologous polypeptides from other species, or for purification ofpolypeptides of the present invention.

The isolated nucleic acids and polypeptides of the present invention canbe used over a broad range of plant types, particularly monocots such asthe species of the family Gramineae including Hordeum, Secale, Oryza,Triticum, Sorghum (e.g., S. bicolor) and Zea (e.g., Z. mays), and dicotssuch as Glycine.

The isolated nucleic acid and proteins of the present invention can alsobe used in species from the genera: Cucurbita, Rosa, Vitis, Juglans,Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna,Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis,Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis,Cucumis, Browallia, Pisum, Phaseolus, Lolium, and Avena.

Nucleic Acids

The present invention provides, among other things, isolated nucleicacids of RNA, DNA, and analogs and/or chimeras thereof, comprising apolynucleotide of the present invention.

A polynucleotide of the present invention is inclusive of those in Table1 and:

(a) an isolated polynucleotide encoding a polypeptide of the presentinvention such as those referenced in Table 1, including exemplarypolynucleotides of the present invention;

(b) an isolated polynucleotide which is the product of amplificationfrom a plant nucleic acid library using primer pairs which selectivelyhybridize under stringent conditions to loci within a polynucleotide ofthe present invention;

(c) an isolated polynucleotide which selectively hybridizes to apolynucleotide of (a) or (b);

(d) an isolated polynucleotide having a specified sequence identity withpolynucleotides of (a), (b) or (c);

(e) an isolated polynucleotide encoding a protein having a specifiednumber of contiguous amino acids from a prototype polypeptide, whereinthe protein is specifically recognized by antisera elicited bypresentation of the protein and wherein the protein does not detectablyimmunoreact to antisera which has been fully immunosorbed with theprotein;

(f) complementary sequences of polynucleotides of (a), (b), (c), (d) or(e);

(g) an isolated polynucleotide comprising at least a specific number ofcontiguous nucleotides from a polynucleotide of (a), (b), (c), (d), (e)or (f);

(h) an isolated polynucleotide from a full-length enriched cDNA libraryhaving the physico-chemical property of selectively hybridizing to apolynucleotide of (a), (b), (c), (d), (e), (f) or (g); and

(i) an isolated polynucleotide made by the process of: 1) providing afull-length enriched nucleic acid library, 2) selectively hybridizingthe polynucleotide to a polynucleotide of (a), (b), (c), (d), (e), (f),(g) or (h), thereby isolating the polynucleotide from the nucleic acidlibrary.

A. Polynucleotides Encoding A Polypeptide of the Present Invention

As indicated in (a), above, the present invention provides isolatednucleic acids comprising a polynucleotide of the present invention,wherein the polynucleotide encodes a polypeptide of the presentinvention. Every nucleic acid sequence herein that encodes a polypeptidealso, by reference to the genetic code, describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine; and UGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Thus, each silent variation of a nucleic acid which encodes apolypeptide of the present invention is implicit in each describedpolypeptide sequence and is within the scope of the present invention.Accordingly, the present invention includes polynucleotides of thepresent invention and polynucleotides encoding a polypeptide of thepresent invention.

B. Polynucleotides Amplified from a Plant Nucleic Acid Library

As indicated in (b), above, the present invention provides an isolatednucleic acid comprising a polynucleotide of the present invention,wherein the polynucleotides are amplified, under nucleic acidamplification conditions, from a plant nucleic acid library. Nucleicacid amplification conditions for each of the variety of amplificationmethods are well known to those of ordinary skill in the art. The plantnucleic acid library can be constructed from a monocot such as a cerealcrop. Exemplary cereals include corn, sorghum, wheat, or rice. The plantnucleic acid library can also be constructed from a dicot such assoybean, alfalfa, or canola. Zea mays lines B73, PHRE1, A632, BMS-P2#10,W23, and Mo17 are known and publicly available. Other publicly known andavailable maize lines can be obtained from the Maize GeneticsCooperation (Urbana, Ill.). Wheat lines are available from the WheatGenetics Resource Center (Manhattan, Kans.).

The nucleic acid library may be a cDNA library, a genomic library, or alibrary generally constructed from nuclear transcripts at any stage ofintron processing. In optional embodiments, the cDNA library isconstructed using an enriched full-length cDNA synthesis method.Examples of such methods include Oligo-Capping (Maruyama and Sugano,(1994) Gene 138:171-174), Biotinylated CAP Trapper (Carninci, et al.,(1996) Genomics 37:327-336), and CAP Retention Procedure (Edery, et al.,(1995) Molecular and Cellular Biology 15:3363-3371). Rapidly growingtissues or rapidly dividing cells are preferred for use as an mRNAsource for construction of a cDNA library. Growth stages of corn aredescribed in “How a Corn Plant Develops,” Special Report No. 48, IowaState University of Science and Technology Cooperative ExtensionService, Ames, Iowa, Reprinted February 1993.

A polynucleotide of this embodiment (or subsequences thereof) can beobtained, for example, by using amplification primers which areselectively hybridized and primer extended, under nucleic acidamplification conditions, to at least two sites within a polynucleotideof the present invention, or to two sites within the nucleic acid whichflank and comprise a polynucleotide of the present invention, or to asite within a polynucleotide of the present invention and a site withinthe nucleic acid which comprises it. Methods for obtaining 5′ and/or 3′ends of a vector insert are well known in the art. See, e.g., RACE(Rapid Amplification of Complementary Ends) as described in Frohman, M.A., in PCR Protocols: A Guide to Methods and Applications, M. A. Innis,D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc.,San Diego), pp. 28-38 (1990)); see, also, U.S. Pat. No. 5,470,722, andCurrent Protocols in Molecular Biology, Unit 15.6, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995); Frohmanand Martin, (1989) Techniques 1:165.

Optionally, the primers are complementary to a subsequence of the targetnucleic acid which they amplify but may have a sequence identity rangingfrom about 85% to 99% relative to the polynucleotide sequence to whichthey are designed to anneal. As those skilled in the art willappreciate, the sites to which the primer pairs will selectivelyhybridize are chosen such that a single contiguous nucleic acid can beformed under the desired nucleic acid amplification conditions. Theprimer length as measured in contiguous nucleotides is selected from thegroup of integers consisting of from at least 15 to 50. Thus, theprimers can be at least 15, 18, 20, 25, 30, 40 or 50 contiguousnucleotides in length. Those of skill will recognize that a lengthenedprimer sequence can be employed to increase specificity of binding(i.e., annealing) to a target sequence. A non-annealing sequence at the5′ end of a primer (a “tail”) can be added, for example, to introduce acloning site at the terminal ends of the amplicon.

The amplification products can be translated using expression systemswell known to those of skill in the art. The resulting translationproducts can be confirmed as polypeptides of the present invention by,for example, assaying for the appropriate catalytic activity (e.g.,specific activity and/or substrate specificity), or verifying thepresence of one or more epitopes which are specific to a polypeptide ofthe present invention. Methods for protein synthesis from PCR-derivedtemplates are known in the art and available commercially. See, e.g.,Amersham Life Sciences, Inc, Catalog '97, p. 354.

C. Polynucleotides Which Selectively Hybridize to a Polynucleotide of(A) or (B)

As indicated in (c), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides selectively hybridize, under selectivehybridization conditions, to a polynucleotide of sections (A) or (B) asdiscussed above. Thus, the polynucleotides of this embodiment can beused for isolating, detecting, and/or quantifying nucleic acidscomprising the polynucleotides of (A) or (B). For example,polynucleotides of the present invention can be used to identify,isolate, or amplify partial or full-length clones in a depositedlibrary. In some embodiments, the polynucleotides are genomic or cDNAsequences isolated or otherwise complementary to a cDNA from a dicot ormonocot nucleic acid library. Exemplary species of monocots and dicotsinclude, but are not limited to: maize, canola, soybean, cotton, wheat,sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley and rice.The cDNA library comprises at least 50% to 95% full-length sequences(for example, at least 50%, 60%, 70%, 80%, 90% or 95% full-lengthsequences). The cDNA libraries can be normalized to increase therepresentation of rare sequences. See, e.g., U.S. Pat. No. 5,482,845.Low stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% to 80% sequence identity and can be employed toidentify orthologous or paralogous sequences.

D. Polynucleotides Having a Specific Sequence Identity with thePolynucleotides of (A), (B) or (C)

As indicated in (d), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides have a specified identity at the nucleotidelevel to a polynucleotide as disclosed above in sections (A), (B) or(C), above. Identity can be calculated using, for example, the BLAST,CLUSTALW, or GAP programs under default conditions. The percentage ofidentity to a reference sequence is at least 60% and, rounded upwards tothe nearest integer, can be expressed as an integer selected from thegroup of integers consisting of from 60 to 99. Thus, for example, thepercentage of identity to a reference sequence can be at least 70%, 75%,80%, 85%, 90% or 95%.

The polynucleotides/polypeptides of the present invention having aspecified sequence identity with a polynucleotide/polypeptide of section(A), (B) or (C) can be of a length (measured in contiguous nucleotidesor amino acids) selected from the group consisting of from 15 to thelength of the polynucleotide/polypeptide of (A), (B) or (C) or anyinteger value in between. For example, the length of the polynucleotidesor polypeptides can be 25, 50, 75, 100, 150, 200, 250, 300, 350, 400,500, 750, 1000, 1250, 1500 or greater.

Optionally, the polynucleotides of this embodiment will encode apolypeptide that will share an epitope with a polypeptide encoded by thepolynucleotides of sections (A), (B) or (C). Thus, these polynucleotidesencode a first polypeptide which elicits production of antiseracomprising antibodies which are specifically reactive to a secondpolypeptide encoded by a polynucleotide of (A), (B) or (C). However, thefirst polypeptide does not bind to antisera raised against itself whenthe antisera has been fully immunosorbed with the first polypeptide.Hence, the polynucleotides of this embodiment can be used to generateantibodies for use in, for example, the screening of expressionlibraries for nucleic acids comprising polynucleotides of (A), (B) or(C), or for purification of, or in immunoassays for, polypeptidesencoded by the polynucleotides of (A), (B) or (C). The polynucleotidesof this embodiment comprise nucleic acid sequences which can be employedfor selective hybridization to a polynucleotide encoding a polypeptideof the present invention.

Screening polypeptides for specific binding to antisera can beconveniently achieved using peptide display libraries. This methodinvolves the screening of large collections of peptides for individualmembers having the desired function or structure. Antibody screening ofpeptide display libraries is well known in the art. The displayedpeptide sequences can be from 3 to 5000 or more amino acids in length,frequently from 5-100 amino acids long, and often from about 8 to 15amino acids long. In addition to direct chemical synthetic methods forgenerating peptide libraries, several recombinant DNA methods have beendescribed. One type involves the display of a peptide sequence on thesurface of a bacteriophage or cell. Each bacteriophage or cell containsthe nucleotide sequence encoding the particular displayed peptidesequence. Such methods are described in PCT Patent Publication Numbers91/17271, 91/18980, 91/19818 and 93/08278. Other systems for generatinglibraries of peptides have aspects of both in vitro chemical synthesisand recombinant methods. See, PCT Patent Publication Numbers 92/05258,92/14843 and 97/20078. See, also, U.S. Pat. Nos. 5,658,754 and5,643,768. Peptide display libraries, vectors, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.).

E. Polynucleotides Encoding a Protein Having a Subsequence from aPrototype Polypeptide and Cross-Reactive to the Prototype Polypeptide

As indicated in (e), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides encode a protein having a subsequence ofcontiguous amino acids from a prototype polypeptide of the presentinvention such as are provided in (a), above. The length of contiguousamino acids from the prototype polypeptide is selected from the group ofintegers consisting of from at least 10 to the number of amino acidswithin the prototype sequence. Thus, for example, the polynucleotide canencode a polypeptide having a subsequence having at least 10, 15, 20,25, 30, 35, 40, 45 or 50, contiguous amino acids from the prototypepolypeptide. Further, the number of such subsequences encoded by apolynucleotide of the instant embodiment can be any integer selectedfrom the group consisting of from 0 to 20, such as 2, 3, 4 or 5. Thesubsequences can be separated by any integer of nucleotides from 0 tothe number of nucleotides in the sequence such as at least 5, 10, 15,25, 50, 100 or 200 nucleotides.

The proteins encoded by polynucleotides of this embodiment, whenpresented as an immunogen, elicit the production of polyclonalantibodies which specifically bind to a prototype polypeptide such asbut not limited to, a polypeptide encoded by the polynucleotide of (a)or (b), above. Generally, however, a protein encoded by a polynucleotideof this embodiment does not bind to antisera raised against theprototype polypeptide when the antisera has been fully immunosorbed withthe prototype polypeptide. Methods of making and assaying for antibodybinding specificity/affinity are well known in the art. Exemplaryimmunoassay formats include ELISA, competitive immunoassays,radioimmunoassays, Western blots, indirect immunofluorescent assays andthe like.

In a preferred assay method, fully immunosorbed and pooled antiserawhich is elicited to the prototype polypeptide can be used in acompetitive binding assay to test the protein. The concentration of theprototype polypeptide required to inhibit 50% of the binding of theantisera to the prototype polypeptide is determined. If the amount ofthe protein required to inhibit binding is less than twice the amount ofthe prototype protein, then the protein is said to specifically bind tothe antisera elicited to the immunogen. Accordingly, the proteins of thepresent invention embrace allelic variants, conservatively modifiedvariants, and minor recombinant modifications to a prototypepolypeptide.

A polynucleotide of the present invention optionally encodes a proteinhaving a molecular weight as the non-glycosylated protein within 20% ofthe molecular weight of the full-length non-glycosylated polypeptides ofthe present invention. Molecular weight can be readily determined bySDS-PAGE under reducing conditions. Optionally, the molecular weight iswithin 15% of a full length polypeptide of the present invention, morepreferably within 10% or 5%, and most preferably within 3%, 2% or 1% ofa full length polypeptide of the present invention.

Optionally, the polynucleotides of this embodiment will encode a proteinhaving a specific enzymatic activity at least 50%, 60%, 80% or 90% of acellular extract comprising the native, endogenous full-lengthpolypeptide of the present invention. Further, the proteins encoded bypolynucleotides of this embodiment will optionally have a substantiallysimilar affinity constant (K_(m)) and/or catalytic activity (i.e., themicroscopic rate constant, k_(cat)) as the native endogenous,full-length protein. Those of skill in the art will recognize thatk_(cat)/K_(m) value determines the specificity for competing substratesand is often referred to as the specificity constant. Proteins of thisembodiment can have a k_(cat)/K_(m) value at least 10% of a full-lengthpolypeptide of the present invention as determined using the endogenoussubstrate of that polypeptide. Optionally, the k_(cat)/K_(m) value willbe at least 20%, 30%, 40%, 50%, and most preferably at least 60%, 70%,80%, 90% or 95% the k_(cat)/K_(m) value of the full-length polypeptideof the present invention. Determination of k_(cat), K_(m), andk_(cat)/K_(m) can be determined by any number of means well known tothose of skill in the art. For example, the initial rates (i.e., thefirst 5% or less of the reaction) can be determined using rapid mixingand sampling techniques (e.g., continuous-flow, stopped-flow, or rapidquenching techniques), flash photolysis, or relaxation methods (e.g.,temperature jumps) in conjunction with such exemplary methods ofmeasuring as spectrophotometry, spectrofluorimetry, nuclear magneticresonance, or radioactive procedures. Kinetic values are convenientlyobtained using a Lineweaver-Burk or Eadie-Hofstee plot.

F. Polynucleotides Complementary to the Polynucleotides of (A)-(E)

As indicated in (f), above, the present invention provides isolatednucleic acids comprising polynucleotides complementary to thepolynucleotides of paragraphs A-E, above. As those of skill in the artwill recognize, complementary sequences base-pair throughout theentirety of their length with the polynucleotides of sections (A)-(E)(i.e., have 100% sequence identity over their entire length).Complementary bases associate through hydrogen bonding in doublestranded nucleic acids. For example, the following base pairs arecomplementary: guanine and cytosine; adenine and thymine; and adenineand uracil.

G. Polynucleotides which are Subsequences of the Polynucleotides of(A)-(F)

As indicated in (g), above, the present invention provides isolatednucleic acids comprising polynucleotides which comprise at least 15contiguous bases from the polynucleotides of sections (A) through (F) asdiscussed above. The length of the polynucleotide is given as an integerselected from the group consisting of from at least 15 to the length ofthe nucleic acid sequence of which the polynucleotide is a subsequence.Thus, for example, polynucleotides of the present invention areinclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50,60, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguousnucleotides in length from the polynucleotides of (A)-(F). Optionally,the number of such subsequences encoded by a polynucleotide of theinstant embodiment can be any integer selected from the group consistingof from 1 to 20, such as 2, 3, 4 or 5. The subsequences can be separatedby any integer of nucleotides from 1 to the number of nucleotides in thesequence such as at least 5, 10, 15, 25, 50, 100 or 200 nucleotides.

Subsequences can be made by in vitro synthetic, in vitro biosynthetic,or in vivo recombinant methods. In optional embodiments, subsequencescan be made by nucleic acid amplification. For example, nucleic acidprimers will be constructed to selectively hybridize to a sequence (orits complement) within, or co-extensive with, the coding region.

A subsequence of the present invention can comprise structuralcharacteristics of the sequence from which it is derived. Alternatively,a subsequence can lack certain structural characteristics of the largersequence from which it is derived such as a poly (A) tail. Optionally, asubsequence from a polynucleotide encoding a polypeptide having at leastone epitope in common with a prototype polypeptide sequence as providedin (a), above, may encode an epitope in common with the prototypesequence. Alternatively, the subsequence may not encode an epitope incommon with the prototype sequence but can be used to isolate the largersequence by, for example, nucleic acid hybridization with the sequencefrom which it is derived. Subsequences can be used to modulate or detectgene expression by introducing into the subsequences compounds whichbind, intercalate, cleave and/or crosslink to nucleic acids. Exemplarycompounds include acridine, psoralen, phenanthroline, naphthoquinone,daunomycin or chloroethylaminoaryl conjugates.

H. Polynucleotides from a Full-Length Enriched cDNA Library Having thePhysico-Chemical Property of Selectively Hybridizing to a Polynucleotideof (A)-(G)

As indicated in (h), above, the present invention provides an isolatedpolynucleotide from a full-length enriched cDNA library having thephysico-chemical property of selectively hybridizing to a polynucleotideof paragraphs (A), (B), (C), (D), (E), (F) or (G) as discussed above.Methods of constructing full-length enriched cDNA libraries are known inthe art and discussed briefly below. The cDNA library comprises at least50% to 95% full-length sequences (for example, at least 50%, 60%, 70%,80%, 90% or 95% full-length sequences). The cDNA library can beconstructed from a variety of tissues from a monocot or dicot at avariety of developmental stages. Exemplary species include maize, wheat,rice, canola, soybean, cotton, sorghum, sunflower, alfalfa, oats, sugarcane, millet, barley and rice. Methods of selectively hybridizing apolynucleotide from a full-length enriched library to a polynucleotideof the present invention are known to those of ordinary skill in theart. Any number of stringency conditions can be employed to allow forselective hybridization. In optional embodiments, the stringency allowsfor selective hybridization of sequences having at least 70%, 75%, 80%,85%, 90%, 95% or 98% sequence identity over the length of the hybridizedregion.

I. Polynucleotide Products Made by a cDNA Isolation Process

As indicated in (I), above, the present invention provides an isolatedpolynucleotide made by the process of: 1) providing a full-lengthenriched nucleic acid library, 2) selectively hybridizing thepolynucleotide to a polynucleotide of paragraphs (A), (B), (C), (D),(E), (F), (G) or (H) as discussed above, and thereby isolating thepolynucleotide from the nucleic acid library. Full-length enrichednucleic acid libraries are constructed as discussed in paragraph (G) andbelow. Selective hybridization conditions are as discussed in paragraph(G). Nucleic acid purification procedures are well known in the art.Purification can be conveniently accomplished using solid-phase methods;such methods are well known to those of skill in the art and kits areavailable from commercial suppliers such as Advanced Biotechnologies(Surrey, UK). For example, a polynucleotide of paragraphs (A)-(H) can beimmobilized to a solid support such as a membrane, bead, or particle.See, e.g., U.S. Pat. No. 5,667,976. The polynucleotide product of thepresent process is selectively hybridized to an immobilizedpolynucleotide and the solid support is subsequently isolated fromnon-hybridized polynucleotides by methods including, but not limited to,centrifugation, magnetic separation, filtration, electrophoresis, andthe like.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a monocot such as corn, rice, or wheat, or a dicot such as soybean.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. A polynucleotideof the present invention can be attached to a vector, adapter, or linkerfor cloning and/or expression of a polynucleotide of the presentinvention. Additional sequences may be added to such cloning and/orexpression sequences to optimize their function in cloning and/orexpression, to aid in isolation of the polynucleotide, or to improve theintroduction of the polynucleotide into a cell. Typically, the length ofa nucleic acid of the present invention less the length of itspolynucleotide of the present invention is less than 20 kilobase pairs,often less than 15 kb, and frequently less than 10 kb. Use of cloningvectors, expression vectors, adapters, and linkers is well known andextensively described in the art. For a description of various nucleicacids see, for example, Stratagene Cloning Systems, Catalogs 1999 (LaJolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '99 (ArlingtonHeights, Ill.).

A Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probeswhich selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. Isolation of RNA andconstruction of cDNA and genomic libraries are well known to those ofordinary skill in the art. See, e.g., Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); andCurrent Protocols in Molecular Biology, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

A1. Full-Length Enriched cDNA Libraries

A number of cDNA synthesis protocols have been described which provideenriched full-length cDNA libraries. Enriched full-length cDNA librariesare constructed to comprise at least 60%, and more preferably at least70%, 80%, 90% or 95% full-length inserts amongst clones containinginserts. The length of insert in such libraries can be at least 2, 3, 4,5, 6, 7, 8, 9, 10 or more kilobase pairs. Vectors to accommodate insertsof these sizes are known in the art and available commercially. See,e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12kb cloning capacity). An exemplary method of constructing a greater than95% pure full-length cDNA library is described by Carninci, et al.,(1996) Genomics, 37:327-336. Other methods for producing full-lengthlibraries are known in the art. See, e.g., Edery, et al., (1995) Mol.Cell. Biol., 15(6):3363-3371; and, PCT Application Number WO 96/34981.

A2 Normalized or Subtracted cDNA Libraries

A non-normalized cDNA library represents the mRNA population of thetissue from which it was made. Since unique clones are out-numbered byclones derived from highly expressed genes, their isolation can belaborious. Normalization of a cDNA library is the process of creating alibrary in which each clone is more equally represented. Construction ofnormalized libraries is described in Ko, (1990) Nucl. Acids. Res.18(19):5705-5711; Patanjali, et al., (1991) Proc. Natl. Acad. U.S.A.88:1943-1947; U.S. Pat. Nos. 5,482,685; 5,482,845 and 5,637,685. In anexemplary method described by Soares, et al., normalization resulted inreduction of the abundance of clones from a range of four orders ofmagnitude to a narrow range of only 1 order of magnitude. Proc. Natl.Acad. Sci. USA 91:9228-9232 (1994).

Subtracted cDNA libraries are another means to increase the proportionof less abundant cDNA species. In this procedure, cDNA prepared from onepool of mRNA is depleted of sequences present in a second pool of mRNAby hybridization. The cDNA:mRNA hybrids are removed and the remainingun-hybridized cDNA pool is enriched for sequences unique to that pool.See, Foote, et al., Plant Molecular Biology: A Laboratory Manual, Clark,Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, (1991) Technique,3(2):58-63; Sive and St. John, (1988) Nucl. Acids Res. 16(22):10937;Current Protocols in Molecular Biology, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995); and, Swaroop, etal., (1991) Nucl. Acids Res., 19(8):1954. cDNA subtraction kits arecommercially available. See, e.g., PCR-Select (Clontech, Palo Alto,Calif.).

To construct genomic libraries, large segments of genomic DNA aregenerated by fragmentation, e.g. using restriction endonucleases, andare ligated with vector DNA to form concatemers that can be packagedinto the appropriate vector. Methodologies to accomplish these ends, andsequencing methods to verify the sequence of nucleic acids, are wellknown in the art. Examples of appropriate molecular biologicaltechniques and instructions sufficient to direct persons of skillthrough many construction, cloning, and screening methodologies arefound in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods inEnzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger andKimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocolsin Molecular Biology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits forconstruction of genomic libraries are also commercially available.

The cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention such as thosedisclosed herein. Probes may be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay, andeither the hybridization or the wash medium can be stringent.

The nucleic acids of interest can also be amplified from nucleic acidsamples using amplification techniques. For instance, polymerase chainreaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes. The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

PCR-based screening methods have been described. Wilfinger, et al.,describe a PCR-based method in which the longest cDNA is identified inthe first step so that incomplete clones can be eliminated from study.BioTechniques, 22(3):481-486 (1997). Such methods are particularlyeffective in combination with a full-length cDNA constructionmethodology, above.

B. Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang, et al., (1979) Meth. Enzymol. 68:90-99; thephosphodiester method of Brown, et al., (1979) Meth. Enzymol.68:109-151; the diethylphosphoramidite method of Beaucage, et al.,(1981) Tetra. Lett. 22:1859-1862; the solid phase phosphoramiditetriester method described by Beaucage and Caruthers, (1981) Tetra.Letts. 22(20):1859-1862, e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter, et al., (1984) Nucleic Acids Res.12:6159-6168; and, the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis generally produces a single-stranded oligonucleotide.This may be converted into double-stranded DNA by hybridization with acomplementary sequence or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is best employed for sequences of about 100bases or less, longer sequences may be obtained by the ligation ofshorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence coding for the desired polypeptide of the present invention,for example a cDNA or a genomic sequence encoding a full-lengthpolypeptide of the present invention, can be used to construct arecombinant expression cassette which can be introduced into the desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences which will direct thetranscription of the polynucleotide in the intended host cell, such aswithin tissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

A plant promoter fragment can be employed which will direct expressionof a polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smaspromoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter,and the GRP1-8 promoter.

Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, and the PPDK promoter which is inducible bylight.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas leaves, roots, fruit, seeds, or flowers. Exemplary promoters includethe anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051), glob-1 promoter, and gamma-zein promoter. The presentinvention provides promoters with expression limited to, or enhanced in,maize silks, including the gl2 promoter (SEQ ID NO: 1 and SEQ ID NO:26). The operation of a promoter may also vary depending on its locationin the genome. Thus, an inducible promoter may become fully or partiallyconstitutive in certain locations.

Both heterologous and non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in recombinantexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter concentration and/or composition of theproteins of the present invention in a desired tissue. Thus, in someembodiments, the nucleic acid construct will comprise a promoter,functional in a plant cell, operably linked to a polynucleotide of thepresent invention. Promoters useful in these embodiments include theendogenous promoters driving expression of a polypeptide of the presentinvention.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up- or down-regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al.,PCT/US93/03868), or isolated promoters can be introduced into a plantcell in the proper orientation and distance from a cognate gene of apolynucleotide of the present invention so as to control the expressionof the gene. Gene expression can be modulated under conditions suitablefor plant growth so as to alter the total concentration and/or alter thecomposition of the polypeptides of the present invention in plant cell.Thus, the present invention provides compositions, and methods formaking, heterologous promoters and/or enhancers operably linked to anative, endogenous (i.e., non-heterologous) form of a polynucleotide ofthe present invention.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

An intron sequence can be added to the 5′ untranslated region or thecoding sequence to increase the amount of the mature message thataccumulates in the cytosol. Inclusion of a spliceable intron in thetranscription unit in both plant and animal expression constructs hasbeen shown to increase gene expression at both the mRNA and proteinlevels up to 1000-fold. Buchman and Berg, (1988) Mol. Cell. Biol.8:4395-4405; Callis, et al., (1987) Genes Dev. 1:1183-1200. Such intronenhancement of gene expression is typically greatest when placed nearthe 5′ end of the transcription unit. Use of maize introns Adh1-S intron1, 2 and 6, the Bronze-1 intron are known in the art. See, generally,The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer,New York (1994). The vector comprising the sequences from apolynucleotide of the present invention will typically comprise a markergene which confers a selectable phenotype on plant cells. Typicalvectors useful for expression of genes in higher plants are well knownin the art and include vectors derived from the tumor-inducing (Ti)plasmid of Agrobacterium tumefaciens described by Rogers, et al., (1987)Meth. in Enzymol. 153:253-277.

A polynucleotide of the present invention can be expressed in eithersense or anti-sense orientation as desired. It will be appreciated thatcontrol of gene expression in either sense or anti-sense orientation canhave a direct impact on the observable plant characteristics. Antisensetechnology can be conveniently used to inhibit gene expression inplants. To accomplish this, a nucleic acid segment from the desired geneis cloned and operably linked to a promoter such that the anti-sensestrand of RNA will be transcribed. The construct is then transformedinto plants and the antisense strand of RNA is produced. In plant cells,it has been shown that antisense RNA inhibits gene expression bypreventing the accumulation of mRNA which encodes the enzyme ofinterest, see, e.g., Sheehy, et al., (1988) Proc. Nat'l. Acad. Sci.(USA) 85:8805-8809; and Hiatt, et al., U.S. Pat. No. 4,801,340.

Another method of suppression is sense suppression (i.e.,co-suppression). Introduction of nucleic acid configured in the senseorientation has been shown to be an effective means by which to blockthe transcription of target genes. For an example of the use of thismethod to modulate expression of endogenous genes, see, Napoli, et al.,(1990) The Plant Cell 2:279-289 and U.S. Pat. No. 5,034,323.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff, et al., (1988)Nature 334:585-591.

A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, et al., (1986) Nucleic Acids Res14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, et al., (1985) Biochimie 67:785-789. Iverson and Dervan alsoshowed sequence-specific cleavage of single-stranded DNA mediated byincorporation of a modified nucleotide which was capable of activatingcleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, et al., (1989) JAm Chem Soc 111:8517-8519, effect covalent crosslinking to a targetnucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. A photoactivatedcrosslinking to single-stranded oligonucleotides mediated by psoralenwas disclosed by Lee, et al., (1988) Biochemistry 27:3197-3203. Use ofcrosslinking in triple-helix forming probes was also disclosed by Home,et al., (1990) J Am Chem Soc 112:2435-2437. Use of N4, N4-ethanocytosineas an alkylating agent to crosslink to single-stranded oligonucleotideshas also been described by Webb and Matteucci, (1986) J Am Chem Soc108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz, et al.,(1991) J. Am. Chem. Soc. 113:4000. Various compounds to bind, detect,label, and/or cleave nucleic acids are known in the art. See, forexample, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648 and5,681,941.

Proteins

The isolated proteins of the present invention comprise a polypeptidehaving at least 10 amino acids from a polypeptide of the presentinvention (or conservative variants thereof) such as those encoded byany one of the polynucleotides of the present invention as discussedmore fully above (e.g., Table 1). The proteins of the present inventionor variants thereof can comprise any number of contiguous amino acidresidues from a polypeptide of the present invention, wherein thatnumber is selected from the group of integers consisting of from 10 tothe number of residues in a full-length polypeptide of the presentinvention. Optionally, this subsequence of contiguous amino acids is atleast 15, 20, 25, 30, 35 or 40 amino acids in length, often at least 50,60, 70, 80 or 90 amino acids in length. Further, the number of suchsubsequences can be any integer selected from the group consisting offrom 1 to 20, such as 2, 3, 4 or 5.

The present invention further provides a protein comprising apolypeptide having a specified sequence identity/similarity with apolypeptide of the present invention. The percentage of sequenceidentity/similarity is an integer selected from the group consisting offrom 50 to 99. Exemplary sequence identity/similarity values include60%, 65%, 70%, 75%, 80%, 85%, 90% and 95%. Sequence identity can bedetermined using, for example, the GAP, CLUSTALW or BLAST programs.

As those of skill will appreciate, the present invention includes, butis not limited to, catalytically active polypeptides of the presentinvention (i.e., enzymes). Catalytically active polypeptides have aspecific activity of at least 20%, 30% or 40%, and preferably at least50%, 60% or 70%, and most preferably at least 80%, 90% or 95% that ofthe native (non-synthetic), endogenous polypeptide. Further, thesubstrate specificity (k_(cat)/K_(m)) is optionally substantiallysimilar to the native (non-synthetic), endogenous polypeptide.Typically, the K_(m) will be at least 30%, 40% or 50%, that of thenative (non-synthetic), endogenous polypeptide; and more preferably atleast 60%, 70%, 80% or 90%. Methods of assaying and quantifying measuresof enzymatic activity and substrate specificity (k_(cat)/K_(m)) are wellknown to those of skill in the art.

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian, or preferably plant cells. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location, and/or time), because they have been geneticallyaltered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter, followed byincorporation into an expression vector. The vector can be suitable forreplication and integration in either prokaryotes or eukaryotes. Typicalexpression vectors contain transcription and translation terminators,initiation sequences, and promoters useful for regulation of theexpression of the DNA encoding a protein of the present invention. Toobtain high level expression of a cloned gene, it is desirable toconstruct expression vectors which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.One of skill would recognize that modifications can be made to a proteinof the present invention without diminishing its biological activity.Some modifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located purification sequences.Restriction sites or termination codons can also be introduced.

Synthesis of Proteins

The proteins of the present invention can be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield, et al., (1963) J. Am. Chem. Soc.85: 2149-2156, and Stewart, et al., Solid Phase Peptide Synthesis, 2nded., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater lengthmay be synthesized by condensation of the amino and carboxy termini ofshorter fragments. Methods of forming peptide bonds by activation of acarboxy terminal end (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

Purification of Proteins

The proteins of the present invention may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced proteins of the present invention can be directly expressed orexpressed as a fusion protein. The recombinant protein is purified by acombination of cell lysis (e.g., sonication, French press) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant protein.

The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including detergent solubilization, selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. The protein may then be isolatedfrom cells expressing the protein and further purified by standardprotein chemistry techniques as described herein. Detection of theexpressed protein is achieved by methods known in the art, including,for example, radioimmunoassays, Western blotting techniques orimmunoprecipitation.

Introduction of Nucleic Acids Into Host Cells

The method of introducing a nucleic acid of the present invention into ahost cell is not critical to the instant invention. Transformation ortransfection methods are conveniently used. Accordingly, a wide varietyof methods have been developed to insert a DNA sequence into the genomeof a host cell to obtain the transcription and/or translation of thesequence to effect phenotypic changes in the organism. Thus, any methodwhich provides for effective introduction of a nucleic acid may beemployed.

A. Plant Transformation

A nucleic acid comprising a polynucleotide of the present invention isoptionally introduced into a plant. Generally, the polynucleotide willfirst be incorporated into a recombinant expression cassette or vector.Isolated nucleic acid acids of the present invention can be introducedinto plants according to techniques known in the art. Techniques fortransforming a wide variety of higher plant species are well known anddescribed in the technical, scientific, and patent literature. See, forexample, Weising, et al., (1988) Ann. Rev. Genet. 22:421-477. Forexample, the DNA construct may be introduced directly into the genomicDNA of the plant cell using techniques such as electroporation,polyethylene glycol (PEG), poration, particle bombardment, silicon fiberdelivery, or microinjection of plant cell protoplasts or embryogeniccallus. See, e.g., Tomes, et al., Direct DNA Transfer into Intact PlantCells Via Microprojectile Bombardment. pp. 197-213 in Plant Cell, Tissueand Organ Culture, Fundamental Methods. eds. O. L. Gamborg and G. C.Phillips, Springer-Verlag Berlin Heidelberg New York, 1995; see, U.S.Pat. No. 5,990,387. The introduction of DNA constructs using PEGprecipitation is described in Paszkowski, et al., (1984) Embo J.3:2717-2722. Electroporation techniques are described in Fromm, et al.,(1985) Proc. Natl. Acad. Sci. (USA) 82:5824. Ballistic transformationtechniques are described in Klein, et al., (1987) Nature 327:70-73.

Agrobacterium tumefaciens-mediated transformation techniques are welldescribed in the scientific literature. See, for example, Horsch, etal., (1984) Science 233:496-498; Fraley, et al., (1983) Proc. Natl.Acad. Sci. (USA) 80:4803; and Plant Molecular Biology: A LaboratoryManual, Chapter 8, Clark, Ed., Springer-Verlag, Berlin (1997). The DNAconstructs may be combined with suitable T-DNA flanking regions andintroduced into a conventional Agrobacterium tumefaciens host vector.The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of the construct and adjacent marker into the plantcell DNA when the cell is infected by the bacteria. See, U.S. Pat. No.5,591,616. Although Agrobacterium is useful primarily in dicots, certainmonocots can be transformed by Agrobacterium. For instance,Agrobacterium transformation of maize is described in U.S. Pat. No.5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, vol. 6, P W J Rigby,Ed., London, Academic Press, 1987; and Lichtenstein, C. P., and Draper,J., In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press,1985), Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)describes the use of A. rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 or pARC16; (2) liposome-mediated DNAuptake (see, e.g., Freeman, et al., (1984) Plant Cell Physiol. 25:1353);(3) the vortexing method (see, e.g., Kindle, (1990) Proc. Natl. Acad.Sci., (USA) 87:1228).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou, et al., (1983) Methods in Enzymology101:433; Hess, (1987) Intern Rev. Cytol. 107:367; Luo, et al., (1988)Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as described by Pena, et al., (1987) Nature 325:274. DNA can alsobe injected directly into the cells of immature embryos and rehydrateddesiccated embryos as described by Neuhaus, et al., (1987) Theor. Appl.Genet. 75:30; and Benbrook, et al., in Proceedings Bio Expo 1986,Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plantviruses that can be employed as vectors are known in the art and includecauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, andtobacco mosaic virus.

B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler,Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson andRoss, Inc. (1977).

Transgenic Plant Regeneration

Plant cells which directly result or are derived from the nucleic acidintroduction techniques can be cultured to regenerate a whole plantwhich possesses the introduced genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium. Plants cells can be regenerated, e.g., from single cells,callus tissue or leaf discs according to standard plant tissue culturetechniques. It is well known in the art that various cells, tissues, andorgans from almost any plant can be successfully cultured to regeneratean entire plant. Plant regeneration from cultured protoplasts isdescribed in Evans, et al., Protoplasts Isolation and Culture, Handbookof Plant Cell Culture, Macmillan Publishing Company, New York, pp.124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts,CRC Press, Boca Raton, pp. 21-73 (1985).

The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, Weissbach and Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). This regeneration and growth processincludes the steps of selection of transformant cells and shoots,rooting the transformant shoots and growth of the plantlets in soil. Formaize cell culture and regeneration see generally, The Maize Handbook,Freeling and Walbot, Eds., Springer, New York (1994); Corn and CornImprovement, 3^(rd) edition, Sprague and Dudley Eds., American Societyof Agronomy, Madison, Wis. (1988). For transformation and regenerationof maize, see, Gordon-Kamm, et al., (1990) The Plant Cell 2:603-618.

The regeneration of plants containing the polynucleotide of the presentinvention and introduced by Agrobacterium from leaf explants can beachieved as described by Horsch, et al., (1985) Science 227:1229-1231.In this procedure, transformants are grown in the presence of aselection agent and in a medium that induces the regeneration of shootsin the plant species being transformed as described by Fraley, et al.,(1983) Proc. Natl. Acad. Sci. USA 80:4803. This procedure typicallyproduces shoots within two to four weeks and these transformant shootsare then transferred to an appropriate root-inducing medium containingthe selective agent and an antibiotic to prevent bacterial growth.Transgenic plants of the present invention may be fertile or sterile.

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced nucleic acid. These seeds can be grownto produce plants with the selected phenotype. Parts obtained from theregenerated plant, such as flowers, seeds, leaves, branches, fruit, andthe like are included in the invention, provided that these partscomprise cells comprising the isolated nucleic acid of the presentinvention. Progeny, variants, and mutants of the regenerated plants arealso included within the scope of the invention, provided that theseparts comprise the introduced nucleic acid sequences. Transgenic plantsexpressing a polynucleotide of the present invention can be screened fortransmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Expression atthe RNA level can be determined initially to identify and quantitateexpression-positive plants. Standard techniques for RNA analysis can beemployed and include PCR amplification assays using oligonucleotideprimers designed to amplify only the heterologous RNA templates andsolution hybridization assays using heterologous nucleic acid-specificprobes. The RNA-positive plants can then be analyzed for proteinexpression by Western immunoblot analysis using the specificallyreactive antibodies of the present invention. In addition, in situhybridization and immunocytochemistry according to standard protocolscan be done using heterologous nucleic acid specific polynucleotideprobes and antibodies, respectively, to localize sites of expressionwithin transgenic tissue. Generally, a number of transgenic lines areusually screened for the incorporated nucleic acid to identify andselect plants with the most appropriate expression profiles.

A preferred embodiment is a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants for alteredexpression of a polynucleotide of the present invention relative to acontrol plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

Modulating Polypeptide Levels and/or Composition

The present invention further provides a method for modulating (i.e.,increasing or decreasing) the concentration or ratio of the polypeptidesof the present invention in a plant or part thereof. Modulation can beeffected by increasing or decreasing the concentration and/or the ratioof the polypeptides of the present invention in a plant. The methodcomprises introducing into a plant cell a recombinant expressioncassette comprising a polynucleotide of the present invention asdescribed above to obtain a transgenic plant cell, culturing thetransgenic plant cell under transgenic plant cell growing conditions,and inducing or repressing expression of a polynucleotide of the presentinvention in the transgenic plant for a time sufficient to modulateconcentration and/or the ratios of the polypeptides in the transgenicplant or plant part.

In some embodiments, the concentration and/or ratios of polypeptides ofthe present invention in a plant may be modulated by altering, in vivoor in vitro, the promoter of a gene to up- or down-regulate geneexpression. In some embodiments, the coding regions of native genes ofthe present invention can be altered via substitution, addition,insertion, or deletion to decrease activity of the encoded enzyme. See,e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868.And in some embodiments, an isolated nucleic acid (e.g., a vector)comprising a promoter sequence is transfected into a plant cell.Subsequently, a plant cell comprising the promoter operably linked to apolynucleotide of the present invention is selected for by means knownto those of skill in the art such as, but not limited to, Southern blot,DNA sequencing, or PCR analysis using primers specific to the promoterand to the gene and detecting amplicons produced therefrom. A plant orplant part altered or modified by the foregoing embodiments is grownunder plant-forming conditions for a time sufficient to modulate theconcentration and/or ratios of polypeptides of the present invention inthe plant. Plant-forming conditions are well known in the art anddiscussed briefly, supra.

In general, concentration or the ratios of the polypeptides is increasedor decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or90% relative to a native control plant, plant part, or cell lacking theaforementioned recombinant expression cassette. Modulation in thepresent invention may occur during and/or subsequent to growth of theplant to the desired stage of development. Modulating nucleic acidexpression temporally and/or in particular tissues can be controlled byemploying the appropriate promoter operably linked to a polynucleotideof the present invention in, for example, sense or antisense orientationas discussed in greater detail, supra. Induction of expression of apolynucleotide of the present invention can also be controlled byexogenous administration of an effective amount of inducing compound.Inducible promoters and inducing compounds which activate expressionfrom these promoters are well known in the art. In preferredembodiments, the polypeptides of the present invention are modulated inmonocots, particularly maize.

UTRs and Codon Preference

In general, translational efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′ UTR) of the RNA. Positive sequence motifs include translationalinitiation consensus sequences (Kozak, (1987) Nucleic Acids Res.15:8125) and the 7-methylguanosine cap structure (Drummond, et al.,(1985) Nucleic Acids Res. 13:7375). Negative elements include stableintramolecular 5′ UTR stem-loop structures (Muesing, et al., (1987) Cell48:691) and AUG sequences or short open reading frames preceded by anappropriate AUG in the 5′ UTR (Kozak, supra, Rao, et al., (1988) Mol.and Cell. Biol. 8:284). Accordingly, the present invention provides 5′and/or 3′ untranslated regions for modulation of translation ofheterologous coding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of thepresent invention can be modified to alter codon usage. Altered codonusage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host such as tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see, Devereaux, etal., (1984) Nucleic Acids Res. 12:387-395) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides that can be used to determine a codon usage frequencycan be any integer from 1 to the number of polynucleotides of thepresent invention as provided herein. Optionally, the polynucleotideswill be full-length sequences. An exemplary number of sequences forstatistical analysis can be at least 1, 5, 10, 20, 50 or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling usingpolynucleotides of the present invention, and compositions resultingtherefrom. Sequence shuffling is described in PCT Publication Number WO97/20078. See, also, Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509. Generally, sequence shuffling provides a means forgenerating libraries of polynucleotides having a desired characteristicwhich can be selected or screened for. Libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides which comprise sequence regions which have substantialsequence identity and can be homologously recombined in vitro or invivo. The population of sequence-recombined polynucleotides comprises asubpopulation of polynucleotides which possess desired or advantageouscharacteristics and which can be selected by a suitable selection orscreening method. The characteristics can be any property or attributecapable of being selected for or detected in a screening system, and mayinclude properties of: an encoded protein, a transcriptional element, asequence controlling transcription, RNA processing, RNA stability,chromatin conformation, translation, or other expression property of agene or transgene, a replicative element, a protein-binding element, orthe like, such as any feature which confers a selectable or detectableproperty. In some embodiments, the selected characteristic will be adecreased K_(m) and/or increased K_(cat) over the wild-type protein asprovided herein. In other embodiments, a protein or polynucleotidegenerated from sequence shuffling will have a ligand binding affinitygreater than the non-shuffled wild-type polynucleotide. The increase insuch properties can be at least 110%, 120%, 130%, 140% or at least 150%of the wild-type value.

Generic and Consensus Sequences

Polynucleotides and polypeptides of the present invention furtherinclude those having: (a) a generic sequence of at least two homologouspolynucleotides or polypeptides, respectively, of the present invention;and (b) a consensus sequence of at least three homologouspolynucleotides or polypeptides, respectively, of the present invention.The generic sequence of the present invention comprises each species ofpolypeptide or polynucleotide embraced by the generic polypeptide orpolynucleotide sequence, respectively. The individual speciesencompassed by a polynucleotide having an amino acid or nucleic acidconsensus sequence can be used to generate antibodies or produce nucleicacid probes or primers to screen for homologs in other species, genera,families, orders, classes, phyla, or kingdoms. For example, apolynucleotide having a consensus sequence from a gene family of Zeamays can be used to generate antibody or nucleic acid probes or primersto other Gramineae species such as wheat, rice, or sorghum.Alternatively, a polynucleotide having a consensus sequence generatedfrom orthologous genes can be used to identify or isolate orthologs ofother taxa. Typically, a polynucleotide having a consensus sequence willbe at least 9, 10, 15, 20, 25, 30 or 40 amino acids in length, or 20,30, 40, 50, 100 or 150 nucleotides in length. As those of skill in theart are aware, a conservative amino acid substitution can be used foramino acids which differ amongst aligned sequences but are from the sameconservative substitution group as discussed above. Optionally, no morethan 1 or 2 conservative amino acids are substituted for each 10 aminoacid length of consensus sequence.

Similar sequences used for generation of a consensus or generic sequenceinclude any number and combination of allelic variants of the same gene,orthologous, or paralogous sequences as provided herein. Optionally,similar sequences used in generating a consensus or generic sequence areidentified using the BLAST program's smallest sum probability (P(N)).Various suppliers of sequence-analysis software are listed in chapter 7of Current Protocols in Molecular Biology, F. M. Ausubel, et al., Eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (Supplement 30). A polynucleotidesequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.1, more preferably less thanabout 0.01, or 0.001, and most preferably less than about 0.0001, or0.00001. Similar polynucleotides can be aligned and a consensus orgeneric sequence generated using multiple sequence alignment softwareavailable from a number of commercial suppliers such as the GeneticsComputer Group's (Madison, Wis.) PILEUP software, Vector NTI's (NorthBethesda, Md.) ALIGNX, or Genecode's (Ann Arbor, Mich.) SEQUENCHER.Conveniently, default parameters of such software can be used togenerate consensus or generic sequences.

Detection of Nucleic Acids

The present invention further provides methods for detecting apolynucleotide of the present invention in a nucleic acid samplesuspected of containing a polynucleotide of the present invention, suchas a plant cell lysate, particularly a lysate of maize. In someembodiments, a cognate gene of a polynucleotide of the present inventionor portion thereof can be amplified prior to the step of contacting thenucleic acid sample with a polynucleotide of the present invention. Thenucleic acid sample is contacted with the polynucleotide to form ahybridization complex. The polynucleotide hybridizes under stringentconditions to a gene encoding a polypeptide of the present invention.Formation of the hybridization complex is used to detect a gene encodinga polypeptide of the present invention in the nucleic acid sample. Thoseof skill will appreciate that an isolated nucleic acid comprising apolynucleotide of the present invention should lack cross-hybridizingsequences in common with non-target genes that would yield a falsepositive result. Detection of the hybridization complex can be achievedusing any number of well-known methods. For example, the nucleic acidsample, or a portion thereof, may be assayed by hybridization formatsincluding but not limited to, solution phase, solid phase, mixed phase,or in situ hybridization assays.

Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, radioisotopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads, fluorescent dyes,radiolabels, enzymes, and calorimetric labels. Other labels includeligands which bind to antibodies labeled with fluorophores,chemiluminescent agents, and enzymes. Labeling the nucleic acids of thepresent invention is readily achieved such as by the use of labeled PCRprimers.

In certain embodiments the nucleic acid sequences of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The combinations generated can include multiplecopies of any one or more of the polynucleotides of interest. Thepolynucleotides of the present invention may be stacked with any gene orcombination of genes to produce plants with a variety of desired traitcombinations, including but not limited to traits desirable for animalfeed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balancedamino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801;5,885,802 and 5,703,409); barley high lysine (Williamson, et al., (1987)Eur. J. Biochem. 165:99-106; and WO 98/20122); and high methionineproteins (Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, etal., (1988) Gene 71:359 and Musumura, et al., (1989) Plant Mol. Biol.12:123)); increased digestibility (e.g., modified storage proteins (U.S.patent application Ser. No. 10/053,410, filed Nov. 7, 2001); andthioredoxins (U.S. patent application Ser. No. 10/005,429, filed Dec. 3,2001)), the disclosures of which are herein incorporated by reference.The polynucleotides of the present invention can also be stacked withtraits desirable for insect, disease or herbicide resistance (e.g.,Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser, et al., (1986) Gene48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol. 24:825);fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence anddisease resistance genes (Jones, et al., (1994) Science 266:789; Martin,et al., (1993) Science 262:1432; Mindrinos, et al., (1994) Cell78:1089); acetolactate synthase (ALS) mutants that lead to herbicideresistance such as the S4 and/or Hra mutations; inhibitors of glutaminesynthase such as phosphinothricin or basta (e.g., bar gene); andglyphosate resistance (EPSPS gene)); and traits desirable for processingor process products such as high oil (e.g., U.S. Pat. No. 6,232,529);modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert,et al., (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe present invention with polynucleotides affecting agronomic traitssuch as male sterility (e.g., see, U.S. Pat. No. 5,583,210), stalkstrength, flowering time, or transformation technology traits such ascell cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364;WO 99/25821), the disclosures of which are herein incorporated byreference.

These stacked combinations can be created by any method, including butnot limited to cross breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences of interest can be driven bythe same promoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of a polynucleotide of interest. This may be accompanied byany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant.

The transformed plants of the invention may be used in a plant breedingprogram. The goal of plant breeding is to combine, in a single varietyor hybrid, various desirable traits. For field crops, these traits mayinclude, for example, resistance to diseases and insects, tolerance toheat and drought, reduced time to crop maturity, greater yield, andbetter agronomic quality. With mechanical harvesting of many crops,uniformity of plant characteristics such as germination and standestablishment, growth rate, maturity, and plant and ear height, isdesirable. Traditional plant breeding is an important tool in developingnew and improved commercial crops. This invention encompasses methodsfor producing a maize plant by crossing a first parent maize plant witha second parent maize plant wherein one or both of the parent maizeplants is a transformed plant displaying enhanced vigor, as describedherein.

Plant breeding techniques known in the art and used in a maize plantbreeding program include, but are not limited to, recurrent selection,bulk selection, mass selection, backcrossing, pedigree breeding, openpollination breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection, doubled haploids, andtransformation. Often combinations of these techniques are used.

The development of maize hybrids in a maize plant breeding programrequires, in general, the development of homozygous inbred lines, thecrossing of these lines, and the evaluation of the crosses. There aremany analytical methods available to evaluate the result of a cross. Theoldest and most traditional method of analysis is the observation ofphenotypic traits. Alternatively, the genotype of a plant can beexamined.

A genetic trait which has been engineered into a particular maize plantusing transformation techniques, could be moved into another line usingtraditional breeding techniques that are well known in the plantbreeding arts. For example, a backcrossing approach is commonly used tomove a transgene from a transformed maize plant to an elite inbred line,and the resulting progeny would then comprise the transgene(s). Also, ifan inbred line was used for the transformation then the transgenicplants could be crossed to a different inbred in order to produce atransgenic hybrid maize plant. As used herein, “crossing” can refer to asimple X by Y cross, or the process of backcrossing, depending on thecontext.

The development of a maize hybrid in a maize plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, which, while different from each other, breedtrue and are highly uniform; and (3) crossing the selected inbred lineswith different inbred lines to produce the hybrids. During theinbreeding process in maize, the vigor of the lines decreases. Vigor isrestored when two different inbred lines are crossed to produce thehybrid. An important consequence of the homozygosity and homogeneity ofthe inbred lines is that the hybrid created by crossing a defined pairof inbreds will always be the same. Once the inbreds that give asuperior hybrid have been identified, the hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

Transgenic plants of the present invention may be used to produce asingle cross hybrid, a three-way hybrid or a double cross hybrid. Asingle cross hybrid is produced when two inbred lines are crossed toproduce the F1 progeny. A double cross hybrid is produced from fourinbred lines crossed in pairs (A×B and C×D) and then the two F1 hybridsare crossed again (A×B)×(C×D). A three-way cross hybrid is produced fromthree inbred lines where two of the inbred lines are crossed (A×B) andthen the resulting F1 hybrid is crossed with the third inbred (A×B)×C.Much of the hybrid vigor and uniformity exhibited by F1 hybrids is lostin the next generation (F2). Consequently, seed produced by hybrids isconsumed rather than planted.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

Example 1

This example describes the construction of a cDNA library.

Total RNA can be isolated from maize tissues with TRIzol Reagent (LifeTechnology Inc. Gaithersburg, Md.) using a modification of the guanidineisothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi(Chomczynski and Sacchi, (1987) Anal. Biochem 162:156). In brief, planttissue samples are pulverized in liquid nitrogen before the addition ofthe TRIzol Reagent, and then further homogenized with a mortar andpestle. Addition of chloroform followed by centrifugation is conductedfor separation of an aqueous phase and an organic phase. The total RNAis recovered by precipitation with isopropyl alcohol from the aqueousphase.

The selection of poly(A)+ RNA from total RNA can be performed usingPolyATact system (Promega Corporation. Madison, Wis.). Biotinylatedoligo(dT) primers are used to hybridize to the 3′ poly(A) tails on mRNA.The hybrids are captured using streptavidin coupled to paramagneticparticles and a magnetic separation stand. The mRNA is then washed athigh stringency conditions and eluted by RNase-free deionized water.

cDNA synthesis and construction of unidirectional cDNA libraries can beaccomplished using the SuperScript Plasmid System (Life Technology Inc.Gaithersburg, Md.). The first strand of cDNA is synthesized by primingan oligo(dT) primer containing a Not I site. The reaction is catalyzedby SuperScript Reverse Transcriptase II at 45° C. The second strand ofcDNA is labeled with alpha-³²P-dCTP and a portion of the reactionanalyzed by agarose gel electrophoresis to determine cDNA sizes. cDNAmolecules smaller than 500 base pairs and unligated adapters are removedby Sephacryl-S400 chromatography. The selected cDNA molecules areligated into pSPORT1 vector in between of Not I and Sal I sites.

Alternatively, cDNA libraries can be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see, Adams, et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2

This method describes construction of a full-length enriched cDNAlibrary.

An enriched full-length cDNA library can be constructed using one of twovariations of the method of Carninci, et al., (1996) Genomics37:327-336. These variations are based on chemical introduction of abiotin group into the diol residue of the 5′ cap structure of eukaryoticmRNA to select full-length first strand cDNA. The selection occurs bytrapping the biotin residue at the cap sites using streptavidin-coatedmagnetic beads followed by RNase I treatment to eliminate incompletelysynthesized cDNAs. Second strand cDNA is synthesized using establishedprocedures such as those provided in Life Technologies' (Rockville, Md.)“SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning” kit.Libraries made by this method have been shown to contain 50% to 70%full-length cDNAs.

The first strand synthesis methods are detailed below. An asteriskdenotes that the reagent was obtained from Life Technologies, Inc.

A. First Strand cDNA Synthesis Method 1 (with Trehalose)

mRNA (10 ug) 25 μl *Not I primer (5 ug) 10 μl *5× 1^(st) strand buffer43 μl *0.1m DTT 20 μl *dNTP mix 10 mm 10 μl BSA 10 ug/μl  1 μl Trehalose(saturated) 59.2 μl   RNase inhibitor (Promega) 1.8 μl  *Superscript IIRT 200 u/μl 20 μl 100% glycerol 18 μl Water  7 μl

The mRNA and Not I primer are mixed and denatured at 65° C. for 10 min.They are then chilled on ice and other components added to the tube.Incubation is at 45° C. for 2 min. Twenty microliters of RT (reversetranscriptase) is added to the reaction and start program on thethermocycler (MJ Research, Waltham, Mass.):

Step 1 45° C. 10 min Step 2 45° C. −0.3° C./cycle, 2 seconds/cycle Step3 go to 2 for 33 cycles Step 4 35° C.  5 min Step 5 45° C.  5 min Step 645° C.  0.2° C./cycle, 1 sec/cycle Step 7 go to 7 for 49 cycles Step 855° C.  0.1° C./cycle, 12 sec/cycle Step 9 go to 8 for 49 cycles Step 1055° C.  2 min Step 11 60° C.  2 min Step 12 go to 11 for 9 times Step 13 4° C. forever Step 14 endB. First Strand cDNA Synthesis Method 2

mRNA (10 μg) 25 μl water 30 μl *Not I adapter primer (5 μg) 10 μl 65° C.for 10 min, chill on ice, then add following reagents, *5× first buffer20 μl *0.1M DTT 10 μl *10 mM dNTP mix  5 μl

Incubate at 45° C. for 2 min, then add 10 μl of *Superscript II RT (200u/μl), start the following program:

Step 1 45° C. for 6 sec, −0.1° C./cycle Step 2 go to 1 for 99 additionalcycles Step 3 35° C. for 5 min Step 4 45° C. for 60 min Step 5 50° C.for 10 min Step 6  4° C. forever Step 7 end

After the 1^(st) strand cDNA synthesis, the DNA is extracted by phenolaccording to standard procedures, and then precipitated in NaOAc andethanol, and stored in −20° C.

C Oxidization of the Diol Group of mRNA for Biotin Labeling

First strand cDNA is spun down and washed once with 70% EtOH. The pelletis resuspended in 23.2 μl of DEPC treated water and put on ice. Prepare100 mM of NalO4 freshly, and then add the following reagents:

mRNA:1^(st) cDNA (start with 20 μg mRNA) 46.4 μl  100 mM NaIO4 (freshlymade) 2.5 μl NaOAc 3M pH 4.5 1.1 μl

To make 100 mM NalO4, use 21.39 μg of NalO4 for 1 μl of water.

Wrap the tube in a foil and incubate on ice for 45 min.

After the incubation, the reaction is then precipitated in:

5M NaCl 10 μl 20% SDS 0.5 μl  isopropanol 61 μl

Incubate on ice for at least 30 min, then spin it down at max speed at4° C. for 30 min and wash once with 70% ethanol and then 80% EtOH.

D. Biotinylation of the mRNA Diol Group

Resuspend the DNA in 110 μl DEPC treated water, then add the followingreagents:

20% SDS 5 μl 2M NaOAc pH 6.1 5 μl 10 mm biotin hydrazide (freshly made)300 μl 

Wrap in a foil and incubate at room temperature overnight.

E. RNase I Treatment

Precipitate DNA in:

5M NaCl 10 μl 2M NaOAc pH 6.1 75 μl biotinylated mRNA:cDNA 420 μl  100%EtOH (2.5 Vol) 1262.5 μl   

(Perform this precipitation in two tubes and split the 420 μl of DNAinto 210 μl each, add 5 μl of 5M NaCl, 37.5 μl of 2M NaOAc pH 6.1, and631.25 μl of 100% EtOH).

Store at −20° C. for at least 30 min. Spin the DNA down at 4° C. atmaximal speed for 30 min. and wash with 80% EtOH twice, then dissolveDNA in 70 μl RNase free water. Pool two tubes and end up with 140 μl.

Add the following reagents:

RNase One 10 U/μl 40 μl 1^(st) cDNA:RNA 140 μl  10X buffer 20 μl

Incubate at 37° C. for 15 min.

Add 5 μl of 40 μg/μl yeast tRNA to each sample for capturing.

F. Full Length 1^(st) cDNA Capturing

Blocking the beads with yeast tRNA:

Beads 1 ml Yeast tRNA 40 μg/μl 5 μl

Incubate on ice for 30 min with mixing, wash 3 times with 1 ml of 2MNaCl, 50 mmEDTA, pH 8.0.

Resuspend the beads in 800% of 2M NaCl, 50 mm EDTA, pH 8.0, add RNase Itreated sample 200 μl, and incubate the reaction for 30 min at roomtemperature. Capture the beads using the magnetic stand, save thesupernatant, and start following washes:

2 washes with 2M NaCl, 50 mm EDTA, pH 8.0, 1 ml each time,

1 wash with 0.4% SDS, 50 μg/ml tRNA,

1 wash with 10 mm Tris-Cl pH 7.5, 0.2 mm EDTA, 10 mm NaCl, 20% glycerol,

1 wash with 50 μg/ml tRNA,

1 wash with 1^(st) cDNA buffer

G. Second Strand cDNA Synthesis

Resuspend the beads in:

*5X first buffer 8 μl *0.1 mM DTT 4 μl *10 mm dNTP mix 8 μl *5X 2ndbuffer 60 μl  *E. coli Ligase 10 U/μl 2 μl *E. coli DNA polymerase 10U/μl 8 μl *E. coli RNaseH 2 U/μl 2 μl P32 dCTP 10 μci/μl 2 μl Or waterup to 300 μl 208 μl 

Incubate at 16° C. for 2 hr with mixing the reaction in every 30 min.

Add 4 μl of T4 DNA polymerase and incubate for additional 5 min at 16°C.

Elute 2^(nd) cDNA from the beads.

Use a magnetic stand to separate the 2^(nd) cDNA from the beads, thenresuspend the beads in 200 μl of water, and then separate again, poolthe samples (about 500 μl),

Add 200 μl of water to the beads, then 200 μl of phenol:chloroform,vortex, and spin to separate the sample with phenol.

Pool the DNA together (about 700 μl) and use phenol to clean the DNAagain, DNA is then precipitated in 2 μg of glycogen and 0.5 vol of 7.5MNH₄OAc and 2 vol of 100% EtOH. Precipitate overnight. Spin down thepellet and wash with 70% EtOH, air-dry the pellet.

DNA 250 μl DNA 200 μl 7.5M NH4OAc 125 μl 7.5M NH4OAc 100 μl 100% EtOH750 μl 100% EtOH 600 μl glycogen 1 μg/μl  2 μl glycogen 1 μg/μl  2 μlH. Sal I Adapter Ligation

Resuspend the pellet in 26 μl of water and use 1 μl for TAE gel.

Set up reaction as following:

2^(nd) strand cDNA 25 μl *5X T4 DNA ligase buffer 10 μl *Sal I adapters10 μl *T4 DNA ligase  5 μl

Mix gently, incubate the reaction at 16° C. overnight.

Add 2 μl of ligase second day and incubate at room temperature for 2 hrs(optional).

Add 50 μl water to the reaction and use 100 μl of phenol to clean theDNA, 90 μl of the upper phase is transferred into a new tube andprecipitate in:

Glycogen 1 μg/μl  2 μl Upper phase DNA 90 μl 7.5M NH4OAc 50 μl 100% EtOH300 μl  precipitate at −20° C. overnight

Spin down the pellet at 4° C. and wash in 70% EtOH, dry the pellet.

I. Not I Digestion

2^(nd) cDNA 41 μl  *Reaction 3 buffer 5 μl *Not I 15 u/μl 4 μl

Mix gently and incubate the reaction at 37° C. for 2 hr.

Add 50 μl of water and 100 μl of phenol, vortex, and take 90 μl of theupper phase to a new tube, then add 50 μl of NH₄₀Ac and 300 μl of EtOH.Precipitate overnight at −20° C.

Cloning, ligation, and transformation are performed per the SuperscriptcDNA synthesis kit.

Example 3

This example describes cDNA sequencing and library subtraction.

Individual colonies can be picked and DNA prepared either by PCR withM13 forward primers and M13 reverse primers, or by plasmid isolation.cDNA clones can be sequenced using M13 reverse primers.

cDNA libraries are plated out on 22×22 cm² agar plate at density ofabout 3,000 colonies per plate. The plates are incubated in a 37° C.incubator for 12-24 hours. Colonies are picked into 384-well plates by arobot colony picker, Q-bot (GENETIX Limited). These plates are incubatedovernight at 37° C. Once sufficient colonies are picked, they are pinnedonto 22×22 cm² nylon membranes using Q-bot. Each membrane holds 9,216 or36,864 colonies. These membranes are placed onto an agar plate with anappropriate antibiotic. The plates are incubated at 37° C. overnight.

After colonies are recovered on the second day, these filters are placedon filter paper prewetted with denaturing solution for four minutes,then incubated on top of a boiling water bath for an additional fourminutes. The filters are then placed on filter paper prewetted withneutralizing solution for four minutes. After excess solution is removedby placing the filters on dry filter papers for one minute, the colonyside of the filters is placed into Proteinase K solution, incubated at37° C. for 40-50 minutes. The filters are placed on dry filter papers todry overnight. DNA is then cross-linked to nylon membrane by UV lighttreatment.

Colony hybridization is conducted as described by Sambrook, et al., (inMolecular Cloning: A laboratory Manual, 2^(nd) Edition). The followingprobes can be used in colony hybridization:

1. First strand cDNA from the same tissue as the library was made fromto remove the most redundant clones.

2. 48-192 most redundant cDNA clones from the same library based onprevious sequencing data.

3. 192 most redundant cDNA clones in the entire maize sequence database.

4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAA AAA AAAAAA AAA, removes clones containing a poly A tail but no cDNA.

5. cDNA clones derived from rRNA.

The image of the autoradiography is scanned into computer and the signalintensity and cold colony addresses of each colony is analyzed.Re-arraying of cold-colonies from 384 well plates to 96 well plates isconducted using Q-bot.

Example 4

This example describes identification of the gene from a computerhomology search.

Gene identities can be determined by conducting BLAST (Basic LocalAlignment Search Tool; Altschul, et al., (1993) J. Mol. Biol.215:403-410; see, also, www.ncbi.nlm.nih.gov/BLAST/) searches underdefault parameters for similarity to sequences contained in the BLAST“nr” database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences are analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN program. The DNA sequences are translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXprogram (Gish and States, (1993) Nature Genetics 3:266-272) provided bythe NCBI. In some cases, the sequencing data from two or more clonescontaining overlapping segments of DNA are used to construct contiguousDNA sequences.

Sequence alignments and percent identity calculations can be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencescan be performed using the Clustal method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method are KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

Example 5

This example describes expression of transgenes in monocot cells.

A transgene can be constructed comprising a cDNA encoding the instantpolypeptides, such as ipt (SEQ ID NO: 2) or ivr2 (SEQ ID NO: 20), insense orientation with respect to a maize silk-preferred promoter, suchas gl2 (SEQ ID NO: 1 or 26), that is located 5′ to the cDNA fragment,and an appropriate termination sequence, such as the 10 kD zein 3′ end,located 3′ to the cDNA fragment. The cDNA fragment of this gene may begenerated by polymerase chain reaction (PCR) of the cDNA clone usingappropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can beincorporated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the digested vector pML103 asdescribed below. Amplification is then performed in a standard PCR. Theamplified DNA is then digested with restriction enzymes NcoI and SmaIand fractionated on an agarose gel. The appropriate band can be isolatedfrom the gel and combined with a 4.9 kb NcoI-SmaI fragment of theplasmid pML103. Plasmid pML103 has been deposited under the terms of theBudapest Treaty at ATCC (American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209), and bears accession numberATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoIpromoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalIfragment from the 3′ end of the maize 10 kD zein gene in the vectorpGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15° C.overnight, essentially as described (Maniatis). The ligated DNA may thenbe used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue;Stratagene). Bacterial transformants can be screened by restrictionenzyme digestion of plasmid DNA and limited nucleotide sequence analysisusing the dideoxy chain termination method (Sequenase DNA SequencingKit; U.S. Biochemical). The resulting plasmid construct would comprise atransgene encoding, in the 5′ to 3′ direction, the maize 27 kD zeinpromoter, a cDNA fragment encoding the instant polypeptides, and the 10kD zein 3′ region.

The transgene described above can then be introduced into corn cells bythe following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu, et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are keptin the dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (Hoechst Ag, Frankfurt, Germany) or equivalent maybe used in transformation experiments in order to provide for aselectable marker. This plasmid contains the Pat gene (see, EuropeanPatent Publication Number 0 242 236) which encodes phosphinothricinacetyl transferase (PAT). The enzyme PAT confers resistance toherbicidal glutamine synthetase inhibitors such as phosphinothricin. Thepat gene in p35S/Ac is under the control of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812) andthe 3′ region of the nopaline synthase gene from the T-DNA of the Tiplasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein, et al., (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 m and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm, et al., (1990) Bio/Technology 8:833-839).

Example 6

This example describes expression of transgenes in dicot cells.

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the 0 subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle,et al., (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embroys may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension are added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7

This example describes expression of a transgene in microbial cells.

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg, et al., (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier, et al., (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 hours at25° C. Cells are then harvested by centrifugation and re-suspended in 50μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One microgram of proteinfrom the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, patent applications, andcomputer programs cited herein are hereby incorporated by reference.

1. A method for enhancing silk exsertion in a Zea mays plant understress, relative to a non-transformed Zea mays plant under stress,comprising transforming said plant or its ancestor with a constructcomprising a silk-specific or silk-preferred promoter of SEQ ID NO: 1 orSEQ ID NO: 26 operably linked to a polynucleotide encoding a polypeptidewhich increases cell division.
 2. The method of claim 1 wherein thepolynucleotide encodes cyclin D.
 3. The method of claim 2 wherein thepolynucleotide encoding cyclin D comprises SEQ ID:
 4. 4. The method ofclaim 1 wherein the polynucleotide encodes a cyclin-dependent kinase. 5.The method of claim 4 wherein the polynucleotide encoding acyclin-dependent kinase comprises SEQ ID NO: 6.